In recent years, various biosensors utilizing a specific catalytic action possessed by enzyme have been developed and in particular, it has been attempted to apply biosensors to the clinical field. In the present days when inspection items and sample numbers are increasing, biosensors which can provide rapid assay with good accuracy have been desired.
Taking a glucose sensor as an example, diabetes has markedly increased nowadays and for measurement and control of blood sugar level in blood, it takes a very long time, since blood is centrifuged and plasma is provided for the measurement as is conventionally done. Thus, a sensor which can make measurement with whole blood is required. As a handy type, there is a stick-like support having provided thereon a carrier containing an enzyme capable of reacting only with glucose and a dye which causes a change upon enzyme reaction or by the product of the enzyme reaction, like a test sheet used for inspection of urine. The stick takes the system that blood is dropped onto the carrier and after a definite period of time, a change of the dye is visually or optically determined. However, interference is serious because of colored matters in blood, resulting in poor accuracy.
Now, a multilayer type analysis carrier as shown in FIG. 1 is proposed (Japanese Utility Model Application Laid-Open No. 54-178495). The carrier has the construction comprising a transparent support 51 having provided thereon, in order, a reagent layer 52, a spreading layer 53, a waterproofing layer 54 and a filtering layer 55. The measurement takes the following system: when a blood sample is dropped from the upside, solid components in blood such as red blood cells, platelets, etc. are removed by the filtering layer 55, the blood uniformly permeates into the spreading layer 53 through a hole 56 in the waterproofing layer and a reaction proceeds in the reagent layer 52. After completion of the reaction, a light is irradiated from the arrow direction through the transparent support 51, whereby a substrate concentration is determined by colorimetry. The system takes a complicated construction as compared to the conventional handy stick-like carrier but its accuracy has improved because blood cells are removed, etc. However, it takes a long time for the permeation of blood and the reaction so that the waterproofing layer 54 that prevents drying of the sample is required. In addition, incubation at a high temperature is required for accelerating the reaction. Thus, the system involves problems that apparatuses and carriers become complicated.
On the other hand, as the system for quantitative assay of a specific component in a sample such as blood, etc. from the living body with high accuracy without performing operations such as dilution, agitation, etc. of the sample solution, a biosensor as shown in FIG. 2 has been proposed (for example, Japanese Patent Application Laid-Open No. 59-166852). The biosensor comprises an insulating base plate 63 having embedded therein an electrode for measurement 64 and a counter electrode 65 made of platinum, etc., having leads 61 and 62, respectively, and the exposed areas of these electrodes are covered with a porous material 66 having carried thereon an oxidoreductase and an electron acceptor. When a sample solution is dropped onto the porous material, the oxidoreductase and the electron acceptor are dissolved in the sample solution, whereby an enzyme reaction proceeds with a substrate in the sample solution and the electron acceptor is reduced. After completion of the reaction, the reduced electron receptor is electrochemically oxidized and a substrate concentration in the sample is determined from a current level for the oxidation obtained in this case. In such a construction, however, the electrodes require operations such as washing, etc., while the porous material can be exchanged for every assay thereby to readily provide for measurement. On the other hand, if it is possible to dispose the apparatus including the electrode system for every measurement, operations for the measurement become extremely simple but from aspects of electrode materials such as platinum, etc., construction and the like, the apparatus is very expensive unavoidably. For the construction of platinum electrodes, the sputtering method or the deposition method can also be used but production costs increase.
As a disposal system including the electrode system, a biosensor described in Japanese Patent Application Laid-Open No. 61-294351 has been proposed. As shown in FIG. 3, in this biosensor, the electrode systems 72 (72'), 73 (73') and 74 (74') composed of carbon, etc. are formed on an insulating base plate 71 by means of screen printing, etc.; after providing an insulating layer 75 thereon, the electrode systems are covered with a porous material 77 having carried thereon an oxidoreductase and an electron acceptor and the whole is integrated with a holding frame 76 and a cover 78. When a sample solution is dropped onto the porous material, the oxidoreductase and the electron acceptor are dissolved in the sample solution, whereby an enzyme reaction proceeds with a substrate in the sample solution and the electron acceptor is reduced. After completion of the reaction, the reduced electron acceptor is electrochemically oxidized and a substrate concentration in the sample is determined from a current level for the oxidation obtained in this case.
In the foregoing conventional construction, the base surface including the electrode system is not always uniformly wetted so that air bubbles remain between the porous material and the base plate, whereby a response current is affected or its reaction rate is reduced in some occasion. Further when an ambient humidity is low, moisture in a sample solution evaporates off during measurement so that a change in response is observed. Furthermore, when a substance that is readily adsorbed to electrodes or a substance such as ascorbic acid or the like that is easily oxidized is present in a sample solution, response of the sensor is affected by such a substance.
Therefore, as sensors for measuring a specific component in a vital sample solution such as blood or the like in a simple and rapid way with high accuracy, sensors which can provide measurement data merely by adding a trace amount of sample solution thereto without diluting or quantitatively determining the sample solution previously are desired. Furthermore, disposable type sensors which are free of operations such as washing, wiping, etc. are desired.